There exist in the art a wide range of techniques for probing molecules, these include techniques such as NMR, ESR, UV and IR spectroscopy in addition to crystallographic techniques, microscopy and electrochemical methods to name just a few.
Many devices can monitor and measure the changes in the structural conformation of molecules. This can be done using techniques which allow interaction to be observed and measured in response to perturbation by solvents, denaturants, temperature, pH or other interacting molecules. Such techniques allow the measurement of thermodynamic parameters such as stability, molecular affinity and associated rate constants for a wide range of molecules such as proteins, DNA, RNA and organic compounds. Perturbation by temperature is particularly useful for thermodynamic evaluations.
One such technique is the measurement of the intrinsic fluorescence of molecules. Fluorescence detection can be carried out using cuvette-based fluorometers or microtiter plate readers with typically 0.2-2 mL and 10-300 μL sample volumes respectively. The temperature of a single sample can be increased or decreased stepwise and the fluorescence measured when each temperature is equilibrated. For example, to determine the thermal stability of a protein or DNA, fluorescence must be measured across a range of typically 10-50 different temperatures in the range from 0 to 100° C. From this the transition mid-point (Tm) temperatures and enthalpy changes upon molecular denaturation can be determined.
The volumes of sample required for cuvette-based fluorometers and microtiter plate readers present two problems. The first is that they require a significant amount of an often limited source of sample. The second is that large volumes of sample take longer to equilibrate to new temperatures than smaller ones. This can be addressed to some degree by increasing the heat-transfer surface-area to volume ratios with a different (thinner) cuvette geometry though this also leads to a loss of fluorescence signal due to the decreased optical path length. However, the slow thermal equilibration times of the water baths, ovens or Peltier blocks used to incubate the sample cuvette or microtitre plate ultimately limits the throughput for measuring a range of increasing or decreasing temperatures to typically no better than 1° C. per minute.
More recently, micro fluidic devices have become available, these use significantly smaller sample volumes (of the order 0.1-10,000 nL) and therefore sample usage is reduced. They can also be devised with much higher heat-transfer surface-area to volume ratios to permit faster transfer of heat to the sample. However, the rate at which a range of temperatures can be sequentially measured at thermal equilibrium is still limited by the equilibration speed of the water baths, ovens or Peltier blocks in contact with the sample container.
One possible solution is the heating of the sample container via an electric current from wires that are in contact with the micro fluidic device surface (for example in WO 03/037514 and WO 03/036302). However, this method still has limitations in terms of the time taken for the wire to reach thermal equilibrium when the temperature is increased or decreased. In most experiments, the temperature of a sample in two or three dimensions is required to be homogeneous.
WO 03/037514 describes the use of two heating elements in contact with a microfluidic channel substrate. Heat transfer through the substrate to the sample in the channel creates a linear temperature gradient within the sample. Such heat transfer by thermal conductance through the substrate and diffusion through the sample requires a period of time for the sample to reach thermal equilibrium. Such a device cannot be used for rapid heating and cooling cycles as the cooling rate of the sample is limited by the cooling rate of the heating elements and local chip substrate. As used herein, with reference to the invention, the term chip is described the “active unit” of the microfluidic device; parallels can be drawn with microchips as the active units of computing devices, the chip of a micro fluidic device is the “active unit” where the sample is located and processed. In general the chip will comprise at least a cavity and means for supplying a sample to the cavity. The use of (electrical) thermal heating elements in WO 03/037514 would require the manufacture of micro fluidic chips with embedded heating elements to ensure good thermal contact. This increases the complexity and cost of manufacture for disposable sample chips.
WO 03/036302 also describes the use of two heating elements in contact with a micro fluidic channel substrate. However, WO 03/036302 additionally describes the use of multiple channels containing samples which flow in a direction from one heating element to another to create a thermal gradient. The thermal gradient can be used to alter the configuration of molecules and is monitored by UV fluorescence. This device has similar disadvantages to WO 03/037514, namely the need to manufacture microfluidic chips with embedded heating elements to ensure good thermal contact. The transfer of heat to the sample by conductance and diffusion is similarly slow, thus limiting the sample flow rate that can be achieved while still creating a thermal gradient across a useful range. Also, the sample cannot be cooled as rapidly as may be desired.
The systems of WO 03/037514 and WO 03/036302, like all of the above methods of sample heating, at all scales, are additionally limited by the need to transfer heat from the edges of the sample container to the centre of the sample; this occurs predominantly by thermal diffusion through the sample. A second disadvantage of these systems is the need to create a microfluidic channel with the heating element in thermal contact with the channel substrate. It is desirable for the micro fluidic chips containing the sample to be disposable due to rapid fouling of the channel by the sample materials. Therefore, the need to embed a thermal heating element within the disposable chip would lead to increased manufacturing complexity and costs.
The use of micro fluidic devices significantly reduces the sample volume and material quantity requirements. However, it would be advantageous to make use of these devices in the controlled rapid and localised heating of samples, to create thermal gradients within a sample, and to simultaneously measure the change in the sample as a result of the heating process at multiple points along the thermal gradient.
Electromagnetic heating of samples has been carried out in cuvette-based and larger sample devices ranging from temperature-jump instruments to domestic and industrial coffee makers. Hoffmann (H. Hofmann, E. Yeager, and J. Stuehr, 1968, Rev. Sci. Instrum., 39, 649) and Eyring (E. M. Eyring and B. C. Bennion, 1968, Ann. Rev. Phys. Chem., 19, 129) described the use of a Q-switched laser shining on a protein sample held, without flow, in a cuvette of 0.05 to 0.5 mm path length and 0.2-20 nL volume. An IR (infra red) laser beam was split to heat both sides of the cuvette evenly in order to create as uniform a thermal excitation as possible over the large probe volumes. A high intensity pulse of IR heating using high power IR lasers was found to raise the temperature of the entire sample very rapidly (ps to ms timescales) and by up to 40° C. Such systems have been used to monitor rapid events such as protein or DNA unfolding. However, such devices cannot be used to maintain a controlled temperature gradient along short microfluidic channels or to create a discrete series of sample plugs along a channel which have sequentially increased temperatures. Neither can they be used to obtain a constant sample temperature. Such gradients or incremental steps of temperature along a short microfluidic channel are required to enable complete equilibrium denaturation curves to be measured rapidly and within a single channel.
IR heating at a single site has also been applied to a liquid sample held between microscope slides (glass plates) and DNA denaturation induced with a temperature shift of up to 60° C. (from 30 to 90° C.) (Baaske et al, 2007, Appl Phys Lett 91, 133901). However, such a system does not permit rapid sample exchange such as is possible in a microfluidic channel. Also the temperature gradient achieved is difficult to control reproducibly in the glass plate system.
The use of UV (ultra violet) or blue lasers or UV-LEDs to induce sample fluorescence in microfluidics devices has also been described (Jaspe & Hagen, 2007 Biophys. J. 91:3415-3424; Schulze et al 2005 Anal. Chem. 77:1325-1329; Lee & Tripathi, 2007). Protein denaturation curves have also been obtained by mixing the protein with varying concentrations of chemical denaturants such as guanidine hydrochloride or urea (Jaspe & Hagen, 2007; Lee & Tripathi, 2007).
US 2005/0164401 describes the use of a light source to heat a sample on a microfluidic chip and to control the temperature of the sample. The technique applies the light source to a stationary sample or at a single position in a flowing sample. This technique cannot create a thermal gradient in a sample along the length of a microfluidic channel.
Accordingly, there is a need in the art to provide a micro fluidic device which overcomes or ameliorates some of the above problems.